• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1084-1091
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• 2013

In this paper, two statistical methods were applied to optimize medium components to improve the production of the milk-clotting enzyme by Bacillus amyloliquefaciens D4. First, wheat bran juice, skim milk powder, and $Na_2HPO_4$ were shown to have significant effects on D4 enzyme production using the Plackett-Burman experimental design. Subsequently, an optimal medium was obtained using the Box-Behnken method, which consisted of 3.31 g/l of skim milk powder, 5.0 g/l of sucrose, 0.1 g/l of $FeSO_4{\cdot}7H_2O$, 0.1 g/l of $MgSO_4{\cdot}7H_2O$, 0.1 g/l of $MnSO_4{\cdot}2H_2O$, 0.1 g/l of $ZnSO_4{\cdot}7H_2O$, 1.52 g/l of $Na_2HPO_4$, and 172.45 g/l of wheat bran juice. With this optimal medium, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 3,326.7 SU/ml after incubation of 48 h, which was 1.76-fold higher than that of the basic medium, showing that the Plackett-Burman design and Box-Behnken response surface method are effective to optimize medium components, and B. amyloliquefaciens D4 possessed a high rennet-producing capacity in the optimal medium.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1250-1256
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• 2011

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.

• Journal of Microbiology and Biotechnology
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• 제21권9호
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• pp.960-967
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• 2011

Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a Plackett-Burman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature ($30^{\circ}C$), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.

• 한국어업기술학회:학술대회논문집
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• 한국어업기술학회 1998년도 수산관련공동학술대회발표요지집
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• pp.222-223
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• 1998

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.443-449
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• 1999

Bacillus subtilis BK-17 which produces a novel protease with fibrinolytic activity was isolated from soybean paste. Bioreactor production of the enzyme was studied in order to optimize fermentation conditions such as medium concentration, pH, agitation speed, and temperature. Under most cultural conditions, enzyme production initially began when the cell growth stopped. The onset of the enzyme production was indicated by rapid increase in both dissolved oxygen (DO) and pH. Two- to three-times more concentrated medium than the flask optimum medium yielded higher enzyme production in the bioreactor fermentation. When the medium pH was controlled constant, pH 6.5 exhibited the highest activity in the range of 6.0 to 7.5, but the activity was similar to the case when the pH was initially adjusted to 7.5 and subsequently maintained within a relatively wide range of 6.4 to 7.8. Agitation speed did not affect the enzyme production with an exception of DO reaching zero. Fermentation time was reduced when temperature increased within the range of $25^{\circ}C$ to$37^{\circ}C$. However, the highest activity, along with the slow decrease of the enzymatic activity after reaching the maximum value, was observed at $25^{\circ}C$. By shifting the temperature from $37^{\circ}C$ to $25^{\circ}C$immediately after DO reached the minimum level, the high enzyme production of 1,100 U/ml along with the short fermentation period of 13 h could be obtained.

• Korean Chemical Engineering Research
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• 제52권3호
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• pp.302-306
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• 2014

목질계 분해효소 활성 증대를 위해 밀짚을 이용한 고체발효에서 주요 발효인자의 최적화를 수행하였다. Trichoderma reesei와 Aspergillus niger를 이용한 혼합배양에서 고체발효에 주요한 영향을 미친다고 알려진 배양온도, pH, 수분함량과 고체기질 크기를 순차적 최적화를 진행하였다. 실험에 적용 된 인자 모두 목질계 분해효소 활성에 유의한 효과를 주었으며, 발효온도 $40^{\circ}C$, pH 7, 수분함량 75%와 고체기질 크기 0.25~0.5 mm가 목질계 분해효소 생산을 위한 최적 조건임을 알 수 있었다. 최적조건 하에서 밀짚을 이용한 고체발효를 수행하였을 때, 효소활성 기준 cellulase 10.3 IU, endoglucanase 100.3 IU, ${\beta}$-glucosidase 22.9 IU와 xylanase 2261.7 IU/g dry material을 배양 96시간에 확인할 수 있었다. 본 결과는 기존 효소활성 대비 각각 72.6, 48.7, 55.2와 51.9% 증가한 수치로 혼합배양과 순차적 최적화를 적용하여 효과적인 목질계 분해효소 활성 증대가 가능함을 확인하였다.

• 미생물학회지
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• 제15권4호
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• pp.145-153
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• 1977

Penicillin amidase (EC 3.5.1.11) was produced by a mutant strain of Bacillius megaterium ATCC 14945. Hydroxylamine assay method for the determination of 6-APT was modified by using "HCl addition techniques" in order to simplify the time consuming orginal assay method without sacrifice of accuracy. Using the new mutant strain, the effects of fermentation conditions on enzyme production were studied.e studied.

• 한국미생물·생명공학회지
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• 제49권2호
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• pp.181-191
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• 2021

The present study addresses isolation, optimization, partial purification, and characterization of a haloalkaline serine protease from a newly isolated haloarchaeal strain isolated from Wadi El Natrun in Egypt. We expected that a two-step sequential statistical approach (one variable at a time, followed by response surface methodology) might maximize the production of the haloalkaline serine protease. The enzyme was partially purified using Hiprep 16/60 sephacryl S-100 HR gel filtration column. Molecular identification revealed the newly isolated haloarchaeon to be Natrialba hulunbeirensis strain WNHS14. Among several tested physicochemical determinants, casamino acids, KCl, and NaCl showed the most significant effects on enzyme production as determined from results of the One-Variable-At-A-time (OVAT) study. The BoxBehnken design localized the optimal levels of the three key determinants; casamino acids, KCl, and NaCl to be 0.5% (w/v), 0.02% (w/v), and 15% (w/v), respectively, obtaining 62.9 U/ml as the maximal amount of protease produced after treatment at 40℃, and pH 9 for 9 days with 6-fold enhancement in yield. The enzyme was partially purified after size exclusion chromatography with specific activity, purification fold, and yield of 1282.63 U/mg, 8.9, and 23%, respectively. The enzyme showed its maximal activity at pH, temperature, and NaCl concentration optima of 10, 75℃, and 2 M, respectively. Phenylmethylsulfonyl fluoride (PMSF, 5 mM) completely inhibited enzyme activity.

• 한국미생물·생명공학회지
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• 제51권1호
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• pp.69-82
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• 2023

Nitrilases are a hydrolase group of enzymes that catalyzes nitrile compounds and produce industrially important organic acids. The current objective is to optimize nitrilase production using statistical methods assisted with artificial intelligence (AI) tool from novel nitrile degrading isolate. A nitrile hydrolyzing bacteria Bacillus subtilis AGAB-2 (GenBank Ascension number- MW857547) was isolated from industrial effluent waste through an enrichment culture technique. The culture conditions were optimized by creating an orthogonal design with 7 variables to investigate the effect of the significant factors on nitrilase activity. On the basis of obtained data, an AI-driven support vector machine was used for the fitted regression, which yielded new sets of predicted responses with zero mean error and reduced root mean square error. The results of the above global optimization were regarded as the theoretical optimal function conditions. Nitrilase activity of 9832 ± 15.3 U/ml was obtained under optimized conditions, which is a 5.3-fold increase in compared to unoptimized (1822 ± 18.42 U/ml). The statistical optimization method involving Plackett Burman Design and Response surface methodology in combination with an AI tool created a better response prediction model with a significant improvement in enzyme production.

• 한국미생물·생명공학회지
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• 제29권2호
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• pp.110-114
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• 2001

가축 사료첨가용 probiotics용 균주를 얻기 위해 특히 내열성이 높아 가공사료에 적합한 Bacillus sp.를 위주로 하여 가축에게 유용한 효소인 phytase를 비롯하여 protease, cellulase, xylanase, amylase의 활성을 모두 나타내는 4-3 균주를 얻어 동정한 결과 Bacillus subtilis로 밝혀졌으며, 이를 B. subtilis 4-3으로 명명하였다. 본 균주를 원료사료에 순수배양하여 사료의 항영양인자인 phytic acid 분해율을 검토한 결과 대두박 및 쌀겨에 있어서는 phytic acid 분해율이 낮았으나, 밀기울의 경우 80.63%로 상대적으로 높은 phytic acid 분해율을 나타내었다. 원료 사료를 이용한 균주의 생산 조건은 대두박 1%(w/v)와 당밀 2%(w/v)가 가장 적합한 균주 생산을 위한 배지조성으로 검토되었다.